To isolate precursor cells derived from rabbit corneal endothelium (CE) and to use them for the treatment of CE deficiency in a rabbit model.A sphere-forming assay was performed to isolate precursor cells from rabbit CE. Immunocytochemistry was used to examine marker expressions of neural and mesenchymal cells in the sphere colonies and their progenies. The pump function of the CE sheet was evaluated by measurement of the potential difference and short circuit current. Precursors obtained from rabbit CE by a sphere-forming assay were injected into the anterior chamber of the eye, after which an eye-down (i.e., CE up) position was maintained for 24 hours to allow attachment by gravitation (sphere eye-down group). The sphere eye-down and control groups, observed for 28 days after surgery, underwent histologic and fluorescence microscopic examinations.Rabbit CE formed primary and secondary sphere colonies. The progeny expressed alpha-smooth muscle actin, nestin, and neural markers and showed a CE-like hexagonal shape and adequate transport activity. Mean corneal thickness in the sphere eye-down group was significantly less than in the other control groups 3, 7, 14, 21, and 28 days (P < 0.05) after surgery. CE-like hexagonal cells were detected on Descemet's membrane, and corneal edema was substantially suppressed. DiI-labeled cells were spread over the rear corneal surface in the sphere eye-down group only.Precursors from rabbit CE were isolated by a sphere-forming assay. Rabbit CE-derived sphere therapy is an effective treatment in a rabbit CE deficiency model.
We determined the plausible functional role of angiopoietin-like protein 2 (Angptl2) in inflammatory corneal hemangiogenesis and lymphangiogenesis in vivo.Corneal hemangiogenesis and lymphangiogenesis were induced by suturing 10-0 nylon 1 mm away from the limbal vessel in Angptl2 knockout and K14-Angptl2 transgenic mice. We analyzed Angptl2 and interleukin 1β (IL-1β) expressions in normal and vascularized corneas by real-time RT-PCR and immunohistochemistry. Corneal hemangiogenic and lymphangiogenic responses, and macrophage infiltration were assessed by immunofluorescent microscopic studies using specific antibodies against CD31, LYVE-1, and F4/80, and compared to their corresponding background. Subconjunctival injection of Angptl2 siRNA to the sutured corneas was also performed.Angptl2 mRNA expression increased markedly in the neovascularized corneas compared to the normal cornea. Angptl2 protein was expressed strongly in the corneal epithelium and stroma of the vascularized cornea. The regions showing hemangiogenesis and lymphangiogenesis were increased significantly in K14-Angptl2 mice and reduced in Angptl2(-/-) mice compared to their corresponding background strains. In contrast to control mice, the number of F4/80-positive cells, as well as the expressions of F4/80 and IL-1β were found to be higher in K14-Angptl2 mice and lower in Angptl2(-/-) mice. Subconjunctival injection of Angptl2 siRNA significantly inhibited hemangiogenesis and lymphangiogenesis in the sutured corneas.Our findings demonstrated Angptl2 to be upregulated in corneal inflammation, and highlight that corneal hemangiogenesis and lymphangiogenesis may be driven by Angptk2 overexpression via macrophage infiltration and IL-1β expression. Angptl2 may be a novel therapeutic target for preventing blindness.
Purpose: To estimate the prevalence of meibomian gland dysfunction (MGD) in the Japanese population. Methods: We undertook a clinical study on the prevalence of MGD in Japan using the same diagnostic criteria as a previous population-based study conducted in Spanish Caucasians. The participants were consecutive patients scheduled for cataract surgery at Inouye Eye Hospital. All participants were aged 50 years or older. Patients completed a symptoms questionnaire and underwent a comprehensive slit-lamp examination. Meibomian gland dysfunction was diagnosed when one or more of the following was present in at least in one eye: absent, viscous, or waxy white secretion upon digital expression; presence of two or more lid margin telangiectases; and/or plugging of two or more gland orifices. Results: The study included 510 patients (205 men and 305 women). Mean participant age was 71.1 ± 8.5 years (range, 50–93 years). The prevalences of symptomatic and total MGD (symptomatic MGD + asymptomatic MGD) were 11.2% and 74.5%, respectively. The prevalence of total MGD increased significantly as participant age increased (P < 0.0001). The ratio of males to females and the prevalence of any systemic disease did not differ between patients who were positive or negative for MGD. For the total MGD group, all slit-lamp findings were more frequent, fluorescein score was higher, tear film breakup time was shorter, and meibo-score was larger, compared to non-MGD patients. Conclusions: Based on the present diagnostic criteria, prevalence of MGD is higher in Tokyo, compared to the Spanish population.
Purpose: Recently, a new commercial test for total tear immunoglobulin E (IgE), based on immunochromatography (Allerwatch), was developed. We examined the relation between the total IgE level in tears and specific serum IgE. Methods: A prospective, nonrandomized, cross-sectional study was conducted in 35 patients with allergic conjunctivitis (allergic group), 30 age- and sex-matched healthy control subjects (control group), and 8 patients with epidemic keratoconjunctivitis. In all subjects, the total tear IgE score was determined with the Allerwatch test (0, 1, and 2), and serum levels of total IgE and specific IgE for 12 inhaled allergens were measured with the Phadezym PRIST and CAP-RAST systems, respectively. Results: The total tear IgE-positive rate was significantly higher in the allergic group than in the control and epidemic keratoconjunctivitis groups (100.0% vs. 0.0% vs. 0.0%; P < 0.00001). In the allergic group, the total tear IgE score was significantly correlated with the log-transformed total serum IgE level (r = 0.61) and with the serum levels of IgE for cedar pollen (r = 0.35), house dust (r = 0.46), Dermatophagoides pteronyssinus (r = 0.49), and acarus (r = 0.36). Multivariate logistic regression analysis showed that the log-transformed total serum IgE level was the only significant predictor of the total tear IgE score (odds ratio = 1.85, P = 0.00008). Conclusions: The total tear IgE score, determined with the Allerwatch test, was correlated with the total and specific serum IgE levels. This rapid test is easy to perform, sensitive, and highly specific for the detection of ocular allergy on an outpatient basis.
Purpose.: The purpose of the present study was to investigate the role of glutathione peroxidase 4 (GPx4) in conjunctival epithelial cells. Methods.: An immortalized human conjunctival epithelial cell line was used. Cells were transfected with catalase, GPx1, GPx4, SOD1, SOD2, or control siRNA. Knockdown was confirmed by RT-PCR and immunoblotting. The cytotoxicity induced by knockdown of these antioxidant enzymes was examined by assay of LDH activity. Furthermore, evaluations of lipid peroxidation, cellular levels of reactive oxygen species, cell proliferation, and apoptosis were conducted in cells treated with GPx4 or control siRNA. In oxidative stress study, cells treated with GPx4 or control siRNA were applied with hydrogen peroxide or ferric sulfide, and their cytotoxicity was evaluated by assay of LDH activity. Results.: Small interfering RNA of catalase, GPx1, GPx4, SOD1, and SOD2 siRNA remarkably inhibited the mRNA and protein expression of each gene. Knockdown of GPx4 and SOD1 but not catalase, GPx1, and SOD2 significantly induced cytotoxicity. Glutathione peroxidase 4 knockdown increased lipid oxidation and reactive oxygen species. The proliferation of GPx4 siRNA-treated cells was reduced compared with control siRNA–treated cells. Moreover, cell death in GPx4 siRNA–treated cells was characterized by positive staining for annexin V. In an oxidation stress study, GPx4 siRNA knockdown enhanced the cytotoxicity induced by hydrogen peroxide or ferric sulfide. Conclusion.: These results suggest that GPx4 is essential for maintaining oxidative homeostasis and keeping defense against oxidative stress in conjunctival epithelial cells.
To introduce photodynamic therapy (PDT) for corneal neovascularization (NV) by polyion complex (PIC) micelles, a novel drug delivery system.Development of specific drug delivery systems to corneal NV sites is an important part of next-generation photodynamic therapy. Nanocarriers consisting of PIC micelles bound by polyethylene glycol (PEG) shell exhibit good stability, high drug-loading capacity, and excellent potential for controlled drug release. Encapsulation of the photosensitizer dendrimer porphyrin (DP) into PIC micelles (DP-micelles) conveys adequate stability and increased photocytotoxicity without compromising photophysical properties of DP stability. We assessed the accumulation of DP-micelles and observed that the DP-micelles were incorporated into the corneal NV area.PIC micelles possess attractive features of selective accumulation at the corneal NV site. PDT using DP-micelles appears to be effective and safe for the treatment of corneal NV.
